The human rhino viruses (HRVs) comprise a large group of closely related picornaviruses which replicate within ciliated epithelial cells and adjacent lymphoid tissues of the upper respiratory tract. Natural infection with HRV effectively stimulates production of local mucosal IgA and systemic IgG antibodies to the virus. The applicant has shown that it is possible to create subgenomic replicon RNAs in which sequence encoding a heterologous protein replaces P1 sequence normally encoding capsid proteins of HRV type 14 (HRV-14). These replicon RNAs are efficiently amplified following delivery to permissive cells and are packaged within the HRV-14 capsid when the P1 precursor protein is expressed in trans within the same cell. These preliminary results provide support for the hypothesis that HRV-14 replicon may serve as an efficient vector for mucosal immunization in humans. The applicant proposes a series of experiments to further refine the requirements for efficient replication and packaging of HRV-14 replicons with consideration of the safety and immunogenicity of such replicons to be evaluated in human volunteers. Specific Aim 1 is to further define a cis-active replication element (CRE-P1) which is located within the P1 region of HRV-14 and which is essential for RNA replication. The investigators plan to map the 5' and 3' limits of the CRE-P1, determine whether it functions as RNA or requires translation, and to examine the requirements for the location and orientation of CRE-P1 within the genome. These results will facilitate optimal design of subsequent HRV-14 replicons. Specific Aim 2 is to construct and evaluate replicon packaging systems utilizing cell lines which constitutively express the P1 precursor protein. Specific Aim 3 is to determine the impact of placement of signal sequences within the replicon polyprotein, and whether HRV-14 replicons can efficiently express glycosylated proteins. Model immunogens will include the respiratory syncytial virus fusion (F) protein and the hepatitis B virus small envelope protein (HBsAg). Specific Aim 4 is to the production of a replicon packaging system under GMP conditions in order to facilitate early phase I testing of candidate replicon vaccines in humans.